Biotechnology promises to provide economical products to save and enhance lives in fields such as medicine, veterinary science, agriculture and horticulture. For example, several genetically engineered pharmaceuticals expressed from animal cells have recently been approved by the Federal Food and Drug Administration. Although this technology is promising and theoretically unlimited in the benefits that it can provide, it is practically limited by challenges in efficiently and economically achieving large scale cell culture production.
One challenge is that many genetically engineered products cannot be produced without culturing mammalian cells. While bacterial and yeast cells are relatively easy to culture, they are not capable of producing many of the complex molecules that are produced naturally by mammals. Moreover, bacterial and yeast cells often cannot properly fold and process foreign proteins. For example bacterial and yeast cells are not capable of glycosylating proteins. Also, these one-celled organisms are undesirable for many large scale biotechnology processes because, as complete organisms, they are generally not equipped to secrete molecules. In order to harvest protein from these organisms, harsh chemicals or mechanical means must be used to fragment the cells. This must be accomplished without disrupting the delicate three dimensional structure of the protein product. Therefore, mammalian cell cultures are preferred for many biotechnology processes.
Although mammalian cells are preferred or required in many cases, mammalian cells have complex requirements because they are not self-supporting organisms like bacteria and yeast. The culture media must be supplemented with a steady stream of nutrients, hormones and other supplements. Moreover, the cells must be handled gently because the delicate, easily ruptured, cell membrane must remain intact for a mammalian cell to survive. Mammalian cells also grow relatively slowly, dividing only every eighteen to forty-eight hours. In addition, most mammalian cells must be attached to a support or substrate similar to their natural conditions in a living organism.
The requirement for attachment presents further challenges in achieving high yields in biotechnology manufacturing plants. Roller bottles with appropriate culture media are typically employed to culture mammalian cells. The standard roller bottle is generally cylindrically shaped and rotatable about its longitudinal axis. The internal surface provides a surface for the cells to attach and grow. Rotating the bottle keeps the internal surfaces wetted with the liquid media to maintain cell life. The use of these devices, media and procedures has been relatively successful for culturing mammalian cells, but yields have generally been relatively low in a manufacturing sense.
Although it has been recognized for quite some time that it is desirable to increase the yield of cells and cell products, certain constraints have limited success in this endeavor. Roller bottle rotation devices are widely provided in standard sizes. These devices are already in place in many laboratories and production facilities and are designed to accept roller bottles of a specific size and shape. Replacing these with custom devices would be expensive and would destroy the standardization that this field has enjoyed. Therefore, it is generally agreed that the outside configuration of roller bottles should remain constant. As such, attempts to increase the number of cells processed per bottle have focused on modifications to the interior surfaces of the roller bottles and improvements in harvesting the cells once they are grown.
Several patents disclose cell culture vessels with modifications to increase the surface area. For example, U.S. Pat. No. 4,962,033 to Serkes et al. describes a roller bottle with corrugations to increase the internal surface area without changing the exterior dimensions of a standard roller bottle. One limitation of this bottle is that cells that attach to the valleys of the corrugations are more difficult to harvest using a scraper or other physical means. This can be a problem if the use of chemical removal agents such as trypsin is not desired or possible due to the effect of the agent on the desired product. U.S. Pat. Nos. 4,004,981 and 4,065,359 to Humi et al. and 4,600,694 disclose scrapers for physically removing cells from disc stacks. Witt (U.S. Pat. No. 4,810,652) discloses a scraper device for harvesting cells from the interior surface of a conventional roller bottle. This device is designed to improve collection of cells but is not compatible with known increased surface area roller bottles such as the corrugated bottle of Serkes et al.
Other approaches to improve yield have included removable cell supports provided within a cell culture vessel. Some of these provide the advantage of adaptation to the standard roller bottle. However, all of the known devices of this type require the use of a tool or require modifications to the construction of the roller bottle, leading to increased initial expense and inefficiencies during use. For example, U.S. Pat. No. 3,941,661 to Notebloom discloses a plastic spiral roller bottle insert that requires a retractor for removal from the roller bottle. A woven sleeve for a roller bottle, disclosed by Mussi et al., increases surface area available for cell growth but does not provide means for physical removal of cells from the sleeve. The sleeve can be removed from the bottle only if the bottle is specially constructed with a removable top portion. U.S. Pat. No. 3,853,712 to House et al. also discloses a flexible ribbon roller bottle insert for increasing surface area. The roller bottle must be dismantled to access the ribbon. The cell culture vessel is provided in two parts sealed with waterproof tape. The tape is removable to disassemble the vessel to access the ribbon for harvesting cells. Once the ribbon is removed, cells may be harvested enzymatically or by such methods as washing, high speed rotation of the vessel, squeegee action or scraping.
One of the problems with some of these approaches is that they greatly increase the cost of each bottle. Since thousands of bottles are used and discarded on a daily basis, even a small increase in cost is quite expensive to a production or research facility. This expense is further aggravated in some cases by the requirement for time consuming and labor intensive procedures. Moreover, many of these known devices do not readily allow physical removal of cells.
Therefore, a need has remained for cell culture devices and methods that are suited for large scale culturing and physical harvesting of mammalian cells. A need has also remained for mammalian cell culture devices that are inexpensive in construction, efficient in use and adaptable to standard laboratory and production scenarios.